The matrix metalloproteinases (MMPs) are a group of zinc enzymes secreted from connective tissue cells and phagocytes. They play a central role in both normal connective tissue remodeling and in accelerated matrix breakdown associated with various connective tissue diseases such as arthritis, gingivitis and tissue ulceration, and in tumor cell invasion and metastasis. Eight members of the MMP family in humans have been identified by cDNA cloning and seven of them have been characterized for their abilities to degrade extracellular matrix macromolecules. This includes collagenases (MMP-1 and MMP-8), gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3 and MMP-10) and a 28-kDa metalloproteinase/Pump- 1/matrilysin (MMP-7). While the synthesis of many MMPs is transcriptionally regulated by various cytokines and growth factors, all of them are secreted from the cells as inactive zymogens (proMMPs). The latter event emphasizes that the extracellular activation processes of the zymogens and their interaction with matrix substrates and endogenous inhibitors are also important regulatory steps in tissue catabolism. The major objective of this proposal is to investigate the activation and the catalytic mechanisms of MMPs in order to understand the precise biological and pathological roles of these enzymes in connective tissue destruction. The catalytic mechanism of MMPs will be investigated using the 28-kDa MMP-3 (stromelysin 1) as a prototype. this will be approached by identification of the side chains involved in binding to tissue inhibitor of metalloproteinases 1 (TIMP-1) and in reaction with alpha2- macroglobulin by a competitive binding "label selection" method. The predicted residues by this study will then be tested by site-directed mutagenesis. In parallel, X-ray crystallographic studies will be conducted in collaboration with Dr. W. Bode at Max-Planck-Institut in Munich. The structural basis for MMP-1 (interstitial collagenase) to express collagenolytic activity will be investigated by homolog-scanning and alanine-scanning mutageneses and by a "differential trace labeling" method. The zymogen activation studies will focus on the kinetic activation analyses of proMMP-2 (72-kDa gelatinase) and proMMP-9 (92-kDa gelatinase) when they are complexed with TIMP-2 and TIMP-1, respectively. Once the detailed knowledge is obtained about the structural and functional relationship of different MMPs to account for their substrate specificities and their catalytic mechanisms, it will be possible to design synthetic inhibitors that can manipulate the unwanted proteolysis of the extracellular matrix that occurs in a variety of connective tissue diseases.